Strain and media
Escherichia coli strain DH5α (Φ80dlacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17(rK-, mK+), supE44, relA1, deoR, Δ(lacZYA-argF)U169) was used in experiments which required a bacterial host. DH5α was grown at 37°C in rich (LB) medium as described in Sambrook et al. [10]. Transformed E. coli cells were maintained on a medium containing 100 mg·L-1 of ampicillin.
H. polymorpha DL-1-356 (leu2) [11] derived from DL1 (ATCC 26012) strain was grown on YPD or synthetic Burkholder medium [12] supplemented with various carbon sources: glucose, methanol and glycerol. Concentration of carbon sources was 1% (w/v or v/v) unless stated otherwise. Leucine was added to final concentration of 40 mg·L-1 if required. For solid media, agar was added to 1.5% (w/v). Yeast cells were grown in Erlenmeyer flasks with continuous shaking (200 rpm) at 37°C.
Mutant isolation
For mutant isolation, cells of the strain H. polymorpha DL-1-356 were cultivated in liquid medium with glucose until midexponential growth phase, washed with distilled water, diluted to 106 cell·mL-1 and irradiated with a dose of UV light that allows 10% cell survival. Then, cells were diluted and plated on the solid Burkholder medium containing methanol (0.5%) and allyl alcohol (0.3 mM). Allyl alcohol was applied as a selective agent; it is oxidized by AOX to acrolein. This compound is toxic for yeast and results in the death of cells with a high AOX activity. On the other hand, AOX activity is essential for growth in medium with methanol as a sole carbon source. Thus, the medium with a mixture of methanol and allyl alcohol will provide a growth of the cells with decreased AOX activity and/or decreased AOX affinity to alcohols. Mutant colonies grown after 21–25 days of incubation were replica-plated into the same medium for confirmatory selection. Mutants with decreased enzyme affinity towards ethanol were screened by a plate colony assay, on the basis of visualization of AOX activity by the rate of hydrogen peroxide formation in reaction with ethanol as monitored by the peroxidative oxidation of o-dianisidine in the presence of peroxidase resulted purple colour of colonies [9]. For this purpose, the plates were incubated at 37°C for 2 days and the colonies formed were replica plated onto agar plates supplemented with glycerol. After 18 h of incubation at 37°C, the plates were overlaid with 9 ml of the alcohol oxidase activity assay mixture, containing 50 mM potassium phosphate buffer (pH 7.5), 0.7% (wt/vol) agar, digitonin (1 mg·mL-1), o-dianisidine (0.5 mg·mL-1), peroxidase Sigma RZ 1.1 (0.13 mg·mL-1), and different (0–15 mM) concentrations of ethanol. The overlying assay mixture was allowed to set, and the plates were incubated at 37°C for up to 1 h. Colonies that stained purple only in the presence of 15 mM ethanol, but not 5 mM or 10 mM, were selected.
Evaluation of kinetic parameters of mutant AOX
AOX affinity towards methanol was evaluated via a kinetic study of enzyme activity in crude cell-free extracts of parental [13] and mutant H. polymorpha strains CA2 and CA4 in the presence of different methanol concentrations (0–100 mM) in the peroxidase reaction with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). KM was calculated from the initial reaction rates, depending on substrate concentration, via the equation:
where V – reaction rate, S – substrate concentration, Vmax and KM – kinetic constants. Constants were calculated with a double-reciprocal method of Lineweaver-Burk [14] using Excel (MS Office pack).
Isolation and analyses of nucleic acids
The 1995 bp coding regions for the AOX was amplified by PCR using the following primers LV1 (5'-CCC AAG CTT ATG GCC ATT CCT GAC GAA TTC-3'); LV2 (5'-CCC AAG CTT TTA GAA TCT GGC AAG TCC G-3') and DNA was extracted from the H. polymorpha DL-1-356, and mutant strains CA2 and CA4. The primers were designed based on the AOX sequence of the H. polymorpha CBS 4732 [15]. The amplified AOX alleles and the vector pUC57, were cut with HindIII and ligated. The resulting plasmids were sequenced. Sequencing of these DNA fragments was performed on both strands. Alignments of putative amino acid sequences were carried out by the programs MultAlin [16] and BOXSHADE 3.21 [17]. The nucleotide sequences of H. polymorpha AOX wild type (DL-1-356), mutant alleles CA2 and CA4 were deposited in GenBank and were assigned the accession nos AM690088, AM690089 and AM690090, respectively.
Molecular biology techniques
Preparations of total DNA from yeast were carried out by using the DNeasy® Tissue Kit (Qiagen, Germany). Plasmid DNA isolations from E. coli were carried out by using NucleoSpin® Plasmid (Macherey-Nagel, Germany).
AOX purification
Highly purified AOX was isolated from a cell-free extract of the isolated mutants, as described elsewhere [8, 18], by means of a two-step ammonium sulphate fractionation (at 30 and 70% of saturation) followed by dialysis and ion exchange chromatography on DEAE-Toyopearl 650 M.
Construction of amperometric biosensor
Amperometric biosensors based on mAOX (CA2) were evaluated using constant-potential amperometry in a three-electrode configuration with a Ag/AgCl/KCl (3 M) reference electrode and a Pt-wire counter electrode. The applied working potential was -50 mV vs Ag/AgCl. Amperometric measurements were carried out using a bipotentiostat (EP 30, Biometra, Göttingen, Germany) connected to a personal computer via a RS232 port for data acquisition. Between experiments, the enzyme electrodes were stored in 20 mM phosphate buffer, pH 7.2, at 4°C.
Bioselective layer of sensor was constructed according to [8]. The structure of the bi-enzyme electrode was configured as HRP/Os-Ap59//AOX(CA2)/CP9 and included two layers: the inner, with horse radish peroxidase (HRP) electrochemically precipitated in the presence of carboxylate-containing polymer modified with an osmium-pyridyl complex (Os-Ap59), and the outer, with mAOX (CA2) immobilized via cathodic precipitation in the presence of an amino-containing polymer (CP9). The Os-containing polymer served as a redox-mediator in the electrochemical reaction. Platinized graphite rods (type RW001, 3.05 mm diameter, Ringsdorff Werke, Bonn, Germany) were used as working electrodes which were sealed in a glass tube using epoxy thus forming disk electrodes.
Determination of biosensors operational stability
The determination of operational stability and inactivation kinetics of the constructed biosensors was performed by using the analyzing system "Olga" (output of sensors to 1 mM ethanol, ejection time 2 min) [19].